Technical Abstract:
Protein-protein interactions are essential for many complex signaling systems that are involved in all levels of cellular processes. Defining, characterizing, and understanding the nature of these protein-protein interactions is a challenging task. Many advanced methodologies, such as tandem affinity purification (TAP), have contributed to dissection of the complexity of multiprotein interactions. Among them, the yeast two-hybrid system has been one of the predominant and powerful tools in this discovery process. The yeast two-hybrid system was developed by Fields and Song in late 1980’s and has been widely used to identify and explore interactions between proteins. This system has recently been adapted to other organisms and to protein-nucleic acid interactions (one- and three- hybrid systems). This chapter focuses on one of two major traditional yeast two-hybrid systems which are based on the separable DNA binding domain (DBD) and transcriptional activation domain (AD) of the yeast Gal 4 transcription factor. The purpose of this chapter is to describe the concepts and principles of yeast two-hybrid system, and the commonly used two-hybrid vectors (like pAS2/pACT2) and yeast strains (like Y190 and Y187). Finally, a hands-on protocol is presented for the construction and testing of fusion plasmids, yeast transformation, and performing library screens.